In fact you do not include any replicates in the graph. You have done the replicates to minimize any errors that might have crept in. For this purpose you should always calculate the mean and the standard deviation. This will tell you which values to consider and which ones to be kept aside.

Hello,

About the IC50 unit: It's μg / mL!

You may include all replicates for different concentrations in the same graph, or have a separate graph for each series of concentrations, both are correct. I advise you to to combine them all in one graph for better visualisation and comparisons.

It is preferred to plot the average IC50 value against each concentration of sample with standard deviation. Therefore you will have a single curve demonstrating the dynamics of scavenging with varying concentration of the sample. Finally you will express the mean IC50 value of the sample with standard error. A second graph is required if you compare the activity of the sample with another one or more samples.

The attached paper has related example-

you should make a graph with IC50 values, and a similar graph by using percent inhibition

In fact you do not include any replicates in the graph. You have done the replicates to minimize any errors that might have crept in. For this purpose you should always calculate the mean and the standard deviation. This will tell you which values to consider and which ones to be kept aside.

Thanks a lot for your replies. I am just concerned about that I will get only one IC50 value from the graph. How can I get the SD for this value?

Dear Fatima:

I believe that you caught me on the wrong foot this time. Well, going by your plan of research, plot all the replicas on different graph. Find out the IC50 values each time and then do the SD. This is very time consuming and if I wer you I would not waste my time doing this. I would rather find out the SD of my replicates and then plot a graph which would give me the most probable IC50.

Hello Fatma, your approach is correct. You need to obtain the IC50 for every replicate, regularly we use triplicates. Then you average the values and obtain the standard deviation. You need to do this for every replicate of your samples and for the control.

As for plotting the results of your assay, you can show an example of a DPPH inhibition curve just for demonstration purposes; but the important results are the IC50, which are better expressed into a table.

Don´t need to estimate the IC50 for all your dilutions. You use serial dilutions as a preliminary test to screen what is the extract concentration that you will use to obtain significant absobance measurements.

Here I attached a link to one of our papers on antioxidant activity for your reference.

Regards.

Article Chemical composition, color, and antioxidant activity of thr...

Thanks a lot @Jai Ghosh and @Rogelio Valadez-Blanco :)

It is very difficult to obtain the actual IC50 experimentally if the sample to be tested has very high antioxidant capacity. As you approach the EC50 experimentally you will get increasing quenching of the sample. I suggest that you put your results from your antioxidant testing into a graph. You will know theoretical IC50 as it is 50% of the reading. If it is absorbance then it will 50% of the absorbance or if you are using chemiluminescence it will be 50% of the maximum light of the no sample control. Once you have this number you should select results that are above the EC50 value. You will need at least 3 points. If a line drawn through these points is perfectly linear then you can extrapolate the real IC50.

I have attached a background document for our ABEL-RAC (relative antioxidant capacity) which gives you an antioxidant score per mg of the dry weight of your sample or uL of liquid. You can obtain an ABEL-RAC score for different ROS challenges not just a general antioxidant score. You and also determine how much of the antioxidant activity is alcohol soluble and how much is water soluble. You can also use this method to quantify synergy between ingredients in a mixture.

We have a template we supply to anyone wanting to generate their own ABEL-RAC mg scores from the different ABEL antioxidant test kits. We also can do contract testing for you.

We will be very pleased to discuss anything with your further. You can also download a lot information from our website www.knightscientific.com.

Jan

I agree with Jan that if the sample has a very high antioxidant activity, the the IC50 values will be a compromised data.

Thanks Jan for your informative reply. You mentioned IC50 and EC50. I found they are used interchangeably for DPPH assay in the literature. Is there a real difference between them?